Grosse R, Rapoport T, Malur J, Fischer J, Repke K R
Biochim Biophys Acta. 1979 Feb 2;550(3):500-14. doi: 10.1016/0005-2736(79)90152-4.
The controlling effect of ATP, K+ and Na+ on the rate of (Na+ + K+)-ATPase inactivation by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-C1) is used for the mathematical modelling of the interaction of the effectors with the enzyme under equilibrium conditions. 1. Of a series of conceivable interaction models, designed without conceptual restrictions to describe the effector control of inactivation kinetics, only one fits the experimental data described in a preceding paper. 2. The model is characterized by the coexistence of two binding sites for ATP and the coexistence of two separate binding sites for K+ and Na+ on the enzyme-ATP complex. On the basis of this model, the effector parameters fitting the experimental data most closely are estimated by means of nonlinear least-squares fits. 3. The apparent dissociation constants for ATP fo the enzyme-ATP complex or of the enzyme-(ATP)2 complex are computed to lie near 0.0024 mM and 0.34 mM, respectively, irrespective of whether K+ and Na+ were absent or K+ and K+ plus Na+, respectively, were present in the experiments. 4. The origin of the high and the low affinity site for binding of ATP to the (Na+ + K+)-ATPase molecule is traced back to the coexistence of two catalytic centres which, although primarily equivalent as to the reactivity of their thiol groups with NBD-C1, are induced into anticooperative communication by ATP binding and thus show an induced geometric asymmetry. 5. On the basis of the interaction model outlined under item 2 the apparent dissociation constant for K+ or Na+ in the (K+ + Na+)-liganded enzyme-ATP complex are computed to be 1.7 mM and 3.5 mM, respectively. 6. The conclusions concerning the coexistence of two primarily equivalent but anticooperatively interacting catalytic centres and the coexistence of two separate ionophoric centres for Na+ and K+ correspond to the appropriate basic postulates of the flip-flop concept of (Na+ + K+)-ATPase mechanism.
利用ATP、K⁺和Na⁺对7-氯-4-硝基苯并-2-恶唑-1,3-二氮杂茂（NBD-C1）使（Na⁺ + K⁺）-ATP酶失活速率的控制作用，对平衡条件下效应物与该酶的相互作用进行数学建模。1. 在一系列为描述效应物对失活动力学的控制而设计、无概念限制的可想象的相互作用模型中，只有一个符合前一篇论文中描述的实验数据。2. 该模型的特征是在酶-ATP复合物上存在两个ATP结合位点以及两个分别独立的K⁺和Na⁺结合位点。基于此模型，通过非线性最小二乘法拟合估算出最符合实验数据的效应物参数。3. 不论实验中是否存在K⁺和Na⁺，还是分别存在K⁺以及K⁺加Na⁺，计算得出酶-ATP复合物或酶-（ATP）₂复合物中ATP的表观解离常数分别接近0.0024 mM和0.34 mM。4. ATP与（Na⁺ + K⁺）-ATP酶分子结合的高亲和力和低亲和力位点的起源可追溯到两个催化中心的共存，这两个催化中心虽然其巯基与NBD-C1的反应性基本相同，但通过ATP结合被诱导进入反协同通讯，从而表现出诱导几何不对称性。5. 根据第2项中概述的相互作用模型，计算得出（K⁺ + Na⁺）配位的酶-ATP复合物中K⁺或Na⁺的表观解离常数分别为1.7 mM和3.5 mM。6. 关于两个基本相同但反协同相互作用的催化中心共存以及两个分别独立的Na⁺和K⁺离子载体中心共存的结论，与（Na⁺ + K⁺）-ATP酶机制的翻转概念的相应基本假设相符。
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